One focus of this project is to elucidate mechanisms involved in and changes consequent to the long-term, repetitive use of drugs. In one study, we used Lewis rats because they show a greater propensity for self-administering cocaine compared to other strains, treated them with cocaine in a long-term but intermittent fashion and found that there was a decrease in the dopamine transporter at 10 days after withdrawal. The change was not evident at earlier times and a withdrawal period was necessary to generate the change as continuous drug administration for the same amount of time did not result in such a decrease. The decrease in transporter was found in the nucleus accumbens, a limbic area associated with addiction, and not in the striatum, an area associated with motoric function. The decrease in transporter was long lasting and still apparent at 60 days after withdrawal. These long term changes are quite intriguing and suggest very long term changes in the brains of human subjects who are withdrawn from cocaine. In order to understand the mechanism of this decrease, we tested whether or not there is a decrease in the messenger RNA for the transporter at the time when the transporter was found to be down-regulated. Because only a small number of the dopaminergic neuronal cell groups project to the accumbens, it was necessary to carry out this study as a light microscopic in situ hybridization investigation as it provided the necessary anatomic resolution to identify the various cell groups of dopaminergic-containing neurons. The in situ hybridization studies revealed that there were different levels of messenger RNA for the dopamine transporter in the different cell groups. In drug-treated and withdrawn animals, it was found that there was a significant reduction in mRNA binding levels in the interfascicular nucleus and in the caudal linear nucleus, but not in the other cell groups of the midbrain. These two cell groups that showed a decrease project to the accumbens. Because of the anatomical correspondence between cells showing a decrease in mRNA and nerve terminal fields showing a decrease in transporter binding, we assume a direct connection between the two and suggest that the down regulation in transporter is due to a decrease in expression of the mRNA. These results indicate that withdrawal from chronic drug administration can result in a series of complicated but long-lasting changes in biochemical parameters in brain. Current directions of this project are aimed at identifying additional proteins or enzymes or transporters that are affected by chronic drug administration.